1. Field of the Invention
1. The present invention relates to wound dressings and more specifically to bioreactor wound dressings which may be used for intentional wounds--surgical incisions and graftings, as well as accidental and unintentional wounds, including cuts, penetrations, burns, sores, ulcers and other deteriorating or damaged skin and flesh wounds.
2. Information Disclosure Statement
To date, no satisfactory dressing has been developed for difficult wounds, such as deep burns or chronic wounds including decubitus ulcers, venous stasis sores, radiation ulcers, pressure sores, ischemic ulcers or diabetic ulcers. Treatment of these wounds remains a very long and expensive process.
It has been found that moist wounds often heal faster. This can be achieved if the desiccation of the wound is prevented by slowing down evaporation of water from the wound. Numerous types of occlusive wound dressings have been developed that are designed to maintain the favorable moist environment in the wound that is deemed essential for the healing process. These dressings promote formation of granular tissue in the wound bed and facilitate migration of epidermal cells. In addition, the occlusive dressings help to control infections. This leads to faster closure of the wound, less pain and less scarring.
One of the problems of occlusive dressings is accumulation of exudate in certain wound types. The exudate interferes with the dressing adhesion and can be a locus of an infection.
This problem can be partly controlled by using hydrogel occlusive dressings, both in the form of a particulate hydrogel dispersion and hydrogel membranes covering the wound. If dehydrated, hydrogel is able to absorb the excess liquid. Various designs of hydrogel-containing absorptive dressings are described in the U.S. Pat. No. 4,909,244, to Quarfoot et al., describes "A wound dressing adapted for preventing of pooling of wound exudate and for promoting healing which is comprised in the following order: (a) a layer consisting essentially of hydrogel for placement on a wound, the hydrogel being characterized as being wound friendly and for absorbing and acting as a reservoir for wound exudate: (b)an intermediate layer disposed over said hydrogel layer. The intermediate layer comprises a tacky hydrogel or hydrocolloid adhesive, the tack hydrogel or hydrocolloid adhesive characterized as having a greater absorbent capacity that does the hydrogel in the hydrogel layer; the underlying hydrogel layer is characterized as being more friendly and consequently more suitable for direct wound contact than the overlying adhesive hydrogel or greater absorbent capacity; the intermediate layer having sufficient moisture permeability there-through to the surface of the dressing; (c) an outer oxygen- and vapor-permeable layer adapted for transpiration of at least a portion of fluid diffusing through the dressing; U.S. Pat. No. 4,979,946, to the inventor Gilman, describes "The creation of an environmental absorbent dressing having an absorbent layer comprising a hydrogel, and a front sheet comprising a film capable of permitting the passage of liquid covering a front surface of the absorbent layer and coated on the front surface thereof with a porous adhesive; U.S. Pat. No. 5,059,424, to the inventor Cartmell, et al, describes "The creation of a wound dressing product which includes a flexible backing member that can be vacuum formed to include a depression. A pressure sensitive adhesive layer extends across the depression of the flexible backing member and a release liner extends over the exposed pressure sensitive adhesive layer and the exposed hydrogel material, which release liner has a selective releasability whereby it can be removed from the wound dressing product intact, leaving a portion of the pressure sensitive adhesive and the hydrogel material exposed; U.S. Pat. No. 5,076,265, to the inventor Wokalek, describes "The creation of a hydrogel sheet for use as a wound dressing with capillaries permitting wound exudate to pass through the sheet without permitting bacteria to infect the wound. The total cross sectional area of the capillaries represents 0.5 to 3.0 percent of the area of the sheet. The sheets do not stick to the wound surface and allow large quantities of wound exudate to be removed from the wound; U.S. Pat. No. 5,106,629, to the inventor Cartmell et al, describes "The creation of a flexible, transparent wound dressing product containing a clear hydrogel material in a gel-like phase. The wound dressing product is comprised of several layers including a wound dressing, a release layer, and a dimensionally stable backing member. The wound dressing is comprised of a thin film transparent layer having an adhesive perimeter portion and a center portion, and a hydrogel material positioned in the center portion of the transparent layer. Since the wound dressing is transparent, a grid pattern may be printed on the thin film transparent layer to permit measurement of a wound. During manufacture, a vacuum pressure is applied to allow temporary access to the center portion of the thin film transparent layer, creating a cavity for insertion of the hydrogel material. The dimensionally stable backing member is then adhesively attached around the perimeter portion of the thin film transparent layer to help the wound dressing maintain its shape. When the wound dressing product is to be applied to a wound site, the release layer is removed, preferable using an extending tab attached thereto, to expose the hydrogen material. The remaining layers of the wound dressing product are then applied to the wound site, with the hydrogel material directly contacting the wound. Once these layers are in place, the dimensionally stable backing member is removed, preferable using and extending tab attached thereto; U.S. Pat. No. 5,112,618, to the inventor Cartmell et al, describes "The creation of a wound dressing product which includes a flexible backing member that can be vacuum formed to include a depression. A pressure sensitive adhesive layer extends across the depression side of the flexible backing member. A hydrogel material is positioned in the depression of the flexible backing member and a release liner extends over the exposed pressure sensitive adhesive layer and the exposed hydrogel material, which release liner has a selective releasability whereby it can be removed from the wound dressing product intact, leaving a portion of the pressure sensitive adhesive and the hydrogel material exposed; U.S. Pat. No. 5,115,801, to the inventor Cartmell et al, describes The creation of a flexible burn dressing product containing a hydrogel material in a gel-like-phase. The burn dressing product is comprised of several layers including a burn dressing and a release layer. The burn dressing is comprised of a bacterial barrier layer coated with a bonding layer, a reticulated layer impregnated with a hydrogel material, and a hydrogel material layer. A dimensionally stable backing member may also be adhesively attached to the bacterial barrier layer to help the burn dressing maintain its shape until it is applied to a patient. When the burn dressing product is to be applied to a burn site, the release layer is removed to expose the hydrogel material layer. The remaining layers of the burn dressing product are then applied to the burn site, with the hydrogel material layer directly contacting the burn. Once these layers are in place, the dimensionally stable backing member is removed; U.S. Pat. No. 5,154,706, to the inventor Cartmell et al, describes "The creation of a wound dressing for a deep wound comprising of hydrogel layer having an upper and lower surface. The hydrogel layer is correspondingly sized to fill the cavity of the deep wound. The wound dressing further comprises a dressing removal layer disposed between the upper surface and the lower surface of the hydrogel layer. The dressing removal layer. extends outwardly from the hydrogel layer so as to form a pull tab which facilitates removal of the hydrogel layer front the deep wound. A method of making a wound dressing for a deep wound also being provided; U.S. Pat. No. 5,204,110, to the inventor Cartmell et al, describes "The creation of a flexible burn dressing product containing a high absorbency hydrogel material in a gel phase. the hydrogel material is formed from a hydrogel composition for use in a wound dressing. The hydrogel composition is formed from a mixture comprising: (a) from about 0% to about 90% by weight polyhydric alcohol selected from the group consisting of polypropylene glycol, polyethylene glycol and glycerine; (b) from about 6% to about 60% by weight aliphatic diisocyanate terminated prepolymer based on polyols containing more than about 40% polyethelyne oxide and having an isocyanate content of about 3% by weight; (c) from about 4% to about 40% by weight polyethelyne oxide based polyamine; (d) up to about 2% by weight sodium chloride; and (e) the balance water. Such a hydrogel material provides a highly absorbent material capable of retaining large amounts of wound exudate, thereby rendering it very suitable for use in wound dressing; U.S. Pat. No. 5,423,736, to the inventor Cartmell et al, describes "The creation of a wound dressing in the form of a gauze or similar absorbent material having dehydrated hydrogel material impregnated therein for absorbing wound exudate. The dehydrated hydrogel material is formed from an aqueous mixture comprising; (a) from about 0% to 90% by weight polyhydric alcohol; (b) from about 6% to about 60% by weight aliphatic diisocyanate terminated prepolymer; (c) from about 4% to about 40% by weight polyethylene oxide based polyamine; (d) 0% to about 2% by weight sodium chloride and (e) the balance water. The present wound dressing is capable of absorbing large amounts of wound exudate without inhibiting the healing of the wound to which it is contacted since it does not adhere or stick to the wound; U.S. Pat. No. 5,423,737, to the inventor Cartmell et al, describes "The creation of a flexible, transparent wound dressing product containing a clear hydrogel material in a gel-like phase. The wound dressing product is comprised of several layers including a wound dressing, removable tab and release liner. The removable tab provides a grippable surface to allow for the removal of the release liner from the wound dressing and to facilitate handling of the wound dressing during application to the wound site. The tab may be comprised of a flat, double coated paper or other suitable material, or a flexible, V shaped member, and is removable by peeling after the wound dressing is applied. The wound dressing comprises a transparent thin film layer, a first adhesive layer, backing layer, second adhesive layer, support layer and a hydrogel material. The transparent layer has a center portion and a perimeter portion. The backing layer, support layer and hydrogel material, which together from a reinforced hydrogel patch, are positioned in the center portion of the transparent layer. Since the wound dressing is transparent, a grid pattern may be printed on the backing layer to permit measurement of a wound. During manufacture, the hydrogel patch is assembled in sheet form and subsequently cut to a desired size and shape; U.S. Pat. No. 5,429,589, to the inventor Cartmell et al, describes "The creation of a wound dressing in the form of a gauze or similar absorbent material having dehydrated hydrogel material impregnated therein for absorbing wound exudate. The dehydrated hydrogel material is formed from an aqueous mixture comprising; (a) from about 0% to 90% by weight polyhydric alcohol; (b) from about 6% to about 60% by weight aliphatic diisocyanate terminated prepolymer; (c) from about 4% to about 40% by weight polyethylene oxide based polyamine; (d) 0% to about 2% by weight sodium chloride and (e) the balance water. The present wound dressing is capable of absorbing large amounts of wound exudate without inhibiting the healing of the wound to which it is contacted since it does not adhere or stick to such a wound; U.S. Pat. No. 5,476,443, as to the inventor Cartmell et al, describes "The creation of a flexible wound dressing product containing a clear hydrogel material in a gel-like phase. The wound dressing product is comprised of several layers including a wound dressing, optional removable tab and optional release liner. The wound dressing comprises a thin film layer, an adhesive layer, porous backing layer, optional support layer and a hydrogel material. The thin film layer has a center portion and a perimeter portion. the backing layer, support layer and hydrogel material, which together from a reinforced hydrogel patch, are positioned in the center portion of the thin film layer. The porous backing layer is formed of a porous material having sufficient porosity that the backing layer can be secured to the hydrogel material without the use of an adhesive. During the manufacture, the hydrogel patch is assembled in sheet form and subsequently cut to a desired size and shape; U.S. Pat. No. 5,489,262, to the inventor Cartmell et al, describes "The creation of a flexible transparent wound dressing product containing a clear hydrogel material in a gel-like phase; U.S. Pat. No. 5,501,661, to the inventor Cartmell et al, describes a flexible wound dressing product containing a clear hydrogel material in a gel-like phase; U.S. Pat. No. 5,531,999, to the inventor Cartmell et al, describes a hydrogel wound dressing substantially in the form of a rope and also in the form or a strand; U.S. Pat. No. 5,674,523, to the inventor Cartmell et al, describes an elongated, self adhesive wound dressing which includes a hydrogel layer secured to a vapor permeable bacterial barrier layer. The vapor permeable barrier layer possess sufficient porosity such that it readily adheres to the hydrogel layer without the need for an adhesive layer. The wound dressing is adapted to be wrapped around a portion of a patient's body and secured without the use of an adhesive and U.S. Pat. No. 5,695,777, to the inventor Donovan et a, describes "The creation of a wound dressing for use with exuding wounds which including (a) an outer vapor permeable layer permitting transpiration of fluid from the dressing; (b) an intermediate layer of hydrogel adapted for absorbing wound exudate; (c) a wound contacting layer for separating the intermediate hydrogel layer from the wound; (d) wicking means associated with the wound contacting layer for conducting exudate from the wound to the hydrogel; and (e) a therapeutic agent retained in the dressing by the wound contacting layer.
Hydrogels are highly permeable for water and allow it to evaporate from the wound faster than many other materials. However, hydrogel occlusive dressings do have several problems. If they are applied to the wound dehydrated, they remove water from the exudate concentrating proteins and other large biomolecules and thus causing protein denaturation and osmotic imbalance. Hydrogels do not perform too well in the absence of an exudate where the hydrogel is supposed to maintain the moist environment of the wound bed. A dehydrated, or partially hydrated hydrogel cannot be a source of moisture. On the contrary, it tends to compete with the cells for water. Hydrogels are also highly permeable for water so that they form, in itself, a poor moisture barrier. Furthermore, a semihydrated hydrogel typically adheres to the wound bed so that the newly formed tissue is injured during the dressing change. It is rarely recognized that the hydrogel properties can favor the healing only if the hydrogel is fully hydrated. From this follows that various types of occlusive dressings are suitable for some types of the wounds and some phases of the healing (i.e., the exudative phase) but they are unsuitable for others. To complicate the dressing selection, the healing often proceeds through various phases with different exudation rates and different dressing requirements. There is currently no dressing that could satisfactorily control the wound bed conditions, particularly due to the variability of these conditions with time. This requires an occasional change of the dressing as the healing progresses. The dressing change disturbs the healing process and exposes the wound to airborne infections.
For that and other reasons, benefits of occlusive dressings are mostly limited to clean wounds, such as surgical incisions or skin donor sites. Occlusive dressings for chronic and deep burn wounds help to form granular tissue and prevent infections. However, they often do not facilitate the epithelization that is essential for the wound closure.
A standard method of healing deep burns is the application of the split-thickness skin grafts. In this technique, a healthy patient skin is excised, sliced and "expanded" into a mesh to cover a large wound area. This method is laborious and, in some cases, it is difficult to excise a sufficient amount of patient's skin without aggravating his/her condition even further. The graft sometimes does not "take" as it has to cope with infection and necrosis of the underlying tissue. One of the disadvantages is also the cross-hatched pattern left by the autologous graft on the newly formed skin.
These limitations of both current synthetic occlusion dressings and autografts lead to continuing search for better "biological" dressings. It is recognized that healing is a very complicated biological process that benefits from presence of a "scaffolding" or matrix of a connective tissue and various biomolecules (such as growth factors, cytokines, glycoaminoglycans and glycoproteins) supporting and controlling the reconstruction of the skin and of underlying tissues. For that reason, numerous methods were developed involving a processed cadaver skin, porcine skin, or tissue-engineered skin substitutes. The cadaver and porcine skin dressings comprise an acellular dermis, i.e. the full-thickness skin from which epidermis and cells were removed, leaving a collagenous matrix. It is believed that some biological activities remain in this processed skin due to residuals of growth factors, glycoproteins and other biomolecules. The collagen matrix is generally not rejected and can serve as a matrix for the cell growth. However, the process of healing and integration of the matrix into the new tissue is slow and sometimes uncertain.
Another approach is seeding of cells into the wound that are capable of forming the new epidermis. The cells used to close the wound are keratinocytes grown as a tissue culture and forming the cultured epidermal graft. Keratinocytes are harvested from a sample of epidermis (obtained by biopsy) and processed by trypsin that destroys desmosomes interconnecting the keratinocytes without harming the cells. The resulting cell dispersion consists mostly of free keratinocytes, with a minor amount of other cells, such as fibroblasts (cells of connective tissue producing collagen), melanocytes (cells producing pigment melanin) and Langerhans cells (specialized macrophage cells). In human epidermis, keratinocytes are fed by products of fibroblasts from the underlying dermis. Consequently, keratinocytes are generally cultivated on a layer of fibroblasts (typically 3T3 mouse fibroblasts). To prevent the fibroblast proliferation, they are irradiated beforehand by a lethal dose of gamma rays. The dying fibroblasts are seeded on the bottom of cultivation flasks filled with a tissue culture medium containing, inter alia, growth factors and stimulants (cholera toxin, insulin, epidermal growth factor etc.). Keratinocytes are then grown on the top of the 3T3 layer.
Only a small portion of keratinocytes harvested from the skin is capable of proliferation (1-10%). The viable cells form colonies that can be harvested and reseeded. About 70% of the reseeded keratinocytes are capable of multiplication and growth. They gradually grow into a confluent layer of keratinocytes interconnected by desmosomes and adhering to the bottom of the flask. As keratinocytes fill the available area, they start growing into a multi-layered stratified sheet. The surface cells (i.e., those facing the solution and far from the flask bottom) terminally differentiate and flatten to resemble corneocytes. New keratinocytes are formed on the bottom of the sheet.
Once the sheet is formed, it can be detached from the bottom by a neutral protease (dispase). The fragile keratinocyte sheet is lifted and transferred onto a support (such as a gauze with Vaseline). The keratinocyte sheet is then applied as a dressing. Those keratinocytes that "take" are attached to the wound bed, proliferate and differentiate into a new epidermis.
This procedure, described by Howard Green, Scientific American, November 1991, p. 96-102, as currently practiced, has a number of problems. The keratinocyte sheet is very fragile. Keratinocytes have to adhere to a suitable location and have to obtain nutrients quickly to remain viable. The direct contact may be difficult to achieve on the irregular wound surface. The adhesion can be prevented by an exudate, bacterial colonization, necrotic tissue or poorly developed granular tissue. The removal of the dressings can damage the newly formed epidermal layer.
The keratinocytes can be obtained from the patient's own epidermis and form a cultured autograft. This is used mainly in treatment of full-thickness skin wounds. This is not very convenient since the cultivation takes approximately 14 to 21 days and is very expensive.
It was found that similar results can be achieved even using keratinocytes from another person, i.e. forming a cultured allograft. (see, for instance: L. A. Y. Duinslaeger et al, Journal of Burn Care & Rehabilitation, November/December 1997, pp. 545-551.) It was observed that Langerhans cells disappear from the tissue culture after about one week. Consequently, even foreign keratinocytes do not elicit a strong rejection if applied as an allograft. Allogeneous keratinocytes do not survive in the wound for long but their presence supports and accelerates healing, probably due to growth factors and cytokines released by them. Keratinocytes for allograft are often harvested from newborn foreskins to maximize the viability and to minimize the probability of transmission of disease.
It was found that keratinocytes can be advantageously grown on a support (such as a polymeric membrane) that can be directly applied to the wound as a dressing. The membrane is advantageously made from a hydrophilic polymer permeable for water that facilitates the exudate removal from the wound (U.S. Pat. No. 5,693,332, to the inventor Hansbrough, describes keratinocytes supported on a hydrophilic membrane). This way, one can avoid the enzymatic detachment of the keratinocyte sheet from the flask and its handling is greatly facilitated. However, this approach has still several problems. The first problem relates to the fact that keratinocytes grown on the polymeric support are nutrified from the solution, i.e. from the other side than in a living skin where they are fed from the dermis underneath. The polymeric membranes used so far are poorly permeable for nutrients and non-permeable for biomolecules, such as growth factors and cytokines. Therefore, the growth on the polymeric support has to be interrupted before or just after the keratinocytes reach confluence so that their upper layers would not interfere with nutrification of the base layer. This limits the efficacy of the keratinocyte cultivation and delivery.
The second problem derives from the requirement that keratinocytes should adhere well to the support to grow and multiply properly. However, as long as they firmly adhere to the polymeric membrane, they are not released to seed the wound. The active biomolecules from the attached keratinocytes can reach the wound bed by diffusion only and their efficacy is thus diminished.
The third problem is that as keratinocytes are applied to the wound, they are transferred from the nutrient solution optimized for their cultivation to the wound bed that is not necessarily a good environment for the keratinocyte growth. It has been reported that less that 10% of autogenous kerationocytes survive this transfer from cultivation media to the wound.
Highly hydrated, well permeable hydrogels are typically a poor support for tissue cultures. Tissue cells do not adhere to them and if they do, they adhere weakly, do not spread and maintain their rounded shape and are unsuitable for their proliferation. This can be explained by highly hydrated. surfaces of such hydrogels that lack hydrophobic attachment points necessary for both attachment of cells, their spreading and adsorption of the underlying proteins. Because of that, keratinocytes have to be cultivated on more. hydrophobic surfaces of partly hydophilized plastics or "marginal" hydrogels with a low water content and low permeability for nutrients. Hydrogels with a low water content are not permeable for many of the nutrients and factors required by keratinocytes, so that they can provide only a limited support for their growth both during the cultivation and after their transfer to the wound. Moreover, hydrogels with a low water content are typically rather rigid so that they cannot conform well to the uneven surface of the wound.
This illustrates the difficulty in meeting all the contradicting requirements with a single polymeric material in the form of a simple polymeric membrane used as the dressing.
Acellular dermis (such as porcine or cadaver dermis) was recently used as the support for the cultured kerationocytes (Hans O. Rennekampff et al, Journal of Burn Care & Rehabilitation, November/December 1997, pp. 535-544; E. Matouskova et al, British Journal of Dermatology 1997; 136:901-907). Keratinocytes adhere firmly to and grow well on the dermis that provides the scaffold, nutrients and growth factors for the keratinocytes as well as occlusive cover and protection to the wound. However, it has also several disadvantages, such as possibility of the infection transmission, possibility of immune reaction, limited range of mechanical and diffusion properties, high handling costs, non-transparency, etc.
Another approach is development of living skin equivalents by tissue engineering methods, such as U.S. Pat. No. Re. 35,399, to the inventor Eisenberg describes "The creation of a composite living skin equivalent comprising of an epidermal layer of cultured keratinocyte cells, al layer of non-porous collagen and a dermal layer of cultured fibroblast cells in a porous cross-linked collagen sponge matrix. Preferable the non-potous collagen is Type 1, Type 3 or mixtures of Types 1 and 3 bovine collagen, which has been pepsin treated. A process for preparing the skin equivalent is described, as well as a test kit for in vitro testing of the skin equivalent. The skin equivalent has use for skin grafting as well as in vitro testing of the effects of various substances on skin." and U.S. Pat. No. 5,460,939, to the inventor Hansbrough et al, describes "The creation of a living skin replacement. in particular, it relates to a biosynthetic dressing material composed of a living stromal tissue prepared from stromal cells such as fibroblasts cultured upon a three dimensional framework and a transitional covering which acts as an epidermal replacement. Such a living skin replacement provides long term biologic coverage of full thickness wound defects. Since human fibroblasts are known to be relatively non-antigenic when transferred to allogeneic hosts, a temporary living skin replacement made up of such cells attached to a transitional covering may replace the use of cadaveric skin allografts for achieving temporary wound closure in cases where the patients lack enough healthy skin for autografts." The disadvantage of such dressings is very high cost and poor shelf life.